Inhibiting DNA methylation alters olfactory extinction but not acquisition learning in Apis cerana and Apis mellifera

DNA methylation plays a key role in invertebrate acquisition and extinction memory. Honey bees have excellent olfactory learning, but the role of DNA methylation in memory formation has, to date, only been studied in Apis mellifera. We inhibited DNA methylation by inhibiting DNA methyltransferase (DNMT) with zebularine (zeb) and studied the resulting effects upon olfactory acquisition and extinction memory in two honey bee species, Apis cerana and A. mellifera. We used the proboscis extension reflex (PER) assay to measure memory. We provide the first demonstration that DNA methylation is also important in the olfactory extinction learning of A. cerana. DNMT did not reduce acquisition learning in either species. However, zeb bidirectionally and differentially altered extinction learning in both species. In particular, zeb provided 1 h before acquisition learning improved extinction memory retention in A. mellifera, but reduced extinction memory retention in A. cerana. The reasons for these differences are unclear, but provide a basis for future studies to explore species-specific differences in the effects of methylation on memory formation.     

Comparative Sucrose Responsiveness in Apis mellifera and A. cerana Foragers

In the European honey bee, Apis mellifera, pollen foragers have a higher sucrose responsiveness than nectar foragers when tested using a proboscis extension response (PER) assay. In addition, Africanized honey bees have a higher sucrose responsiveness than European honey bees. Based on the biology of the Eastern honey bee, A. cerana, we hypothesized that A. cerana should also have a higher responsiveness to sucrose than A. mellifera. To test this hypothesis, we compared the sucrose thresholds of pollen foragers and nectar foragers in both A. cerana and A. mellifera in Fujian Province, China. Pollen foragers were more responsive to sucrose than nectar foragers in both species, consistent with previous studies. However, contrary to our hypothesis, A. mellifera was more responsive than A. cerana. We also demonstrated that this higher sucrose responsiveness in A. mellifera was not due to differences in the colony environment by co-fostering two species of bees in the same mixed-species colonies. Because A. mellifera foragers were more responsive to sucrose, we predicted that their nectar foragers should bring in less concentrated nectar compared to that of A. cerana. However, we found no differences between the two species. We conclude that A. cerana shows a different pattern in sucrose responsiveness from that of Africanized bees. There may be other mechanisms that enable A. cerana to perform well in areas with sparse nectar resources.     

Flower visitation patterns of the coexisting honey bees Apis cerana japonica and Apis mellifera (Hymenoptera: Apidae)

We compared flower visitation patterns of two coexisting honey bees, Apis mellifera Linnaeus and Apis cerana japonica Radoszkowski, on 20 plant species, including three exotics, under natural conditions in Nara, Japan, from April to August 2012. We also measured flower color based on bee color vision (15 flower species), nectar volume (nine species) and nectar concentration (eight species). Flowers colored white, pink, red, purple and cream were classified as bee‐blue‐green, and yellow was classified as bee‐green. Apis cerana visited 14 plant species and A. mellifera visited 11. Although the two Apis species are similar in morphology, they visited different plants: in particular, A. cerana visited native plant species more often than did A. mellifera. Both A. mellifera and A. cerana visited not only nectariferous flowers but also those with no nectar. We also found different visitation patterns between A. cerana and A. mellifera: Apis cerana more often visited flowers with smaller color angle (bee‐blue‐green), lower chroma and higher brightness, and flowers secreting nectars of higher concentration and smaller volume than did A. mellifera.     

Uncovering the novel characteristics of Asian honey bee,Apis cerana,by whole genome sequencing

Background

The honey bee is an important model system for increasing understanding of molecular and neural mechanisms underlying social behaviors relevant to the agricultural industry and basic science. The western honey bee, Apis mellifera, has served as a model species, and its genome sequence has been published. In contrast, the genome of the Asian honey bee, Apis cerana, has not yet been sequenced. A. cerana has been raised in Asian countries for thousands of years and has brought considerable economic benefits to the apicultural industry. A cerana has divergent biological traits compared to A. mellifera and it has played a key role in maintaining biodiversity in eastern and southern Asia. Here we report the first whole genome sequence of A. cerana.

Results

Using de novo assembly methods, we produced a 238 Mbp draft of the A. cerana genome and generated 10,651 genes. A.cerana-specific genes were analyzed to better understand the novel characteristics of this honey bee species. Seventy-two percent of the A. cerana-specific genes had more than one GO term, and 1,696 enzymes were categorized into 125 pathways. Genes involved in chemoreception and immunity were carefully identified and compared to those from other sequenced insect models. These included 10 gustatory receptors, 119 odorant receptors, 10 ionotropic receptors, and 160 immune-related genes.

Conclusions

This first report of the whole genome sequence of A. cerana provides resources for comparative sociogenomics, especially in the field of social insect communication. These important tools will contribute to a better understanding of the complex behaviors and natural biology of the Asian honey bee and to anticipate its future evolutionary trajectory.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-16-1) contains supplementary material, which is available to authorized users.     

Pyrosequencing analysis of the bacterial communities in the guts of honey bees Apis cerana and Apis mellifera in Korea

The bacterial communities in the guts of the adults and larvae of the Asian honey bee Apis cerana and the European honey bee Apis mellifera were surveyed by pyrosequencing the 16S rRNA genes. Most of the gut bacterial 16S rRNA gene sequences were highly similar to the known honey bee-specific ones and affiliated with Pasteurellaceae or lactic acid bacteria (LAB). The numbers of operational taxonomic units (OTUs, defined at 97% similarity) were lower in the larval guts (6 or 9) than in the adult guts (18 or 20), and the frequencies of Pasteurellaceae-related OTUs were higher in the larval guts while those of LAB-related OTUs in the adult guts. The frequencies of Lactococcus, Bartonella, Spiroplasma, Enterobacteriaceae, and Flavobacteriaceae-related OTUs were much higher in A. cerana guts while Bifidobacterium and Lachnospiraceae-related OTUs were more abundant in A. mellfera guts. The bacterial community structures in the midguts and hindguts of the adult honey bees were not different for A. cerana, but significantly different for A. mellifera. The above results substantiated the previous observation that honey bee guts are dominated by several specific bacterial groups, and also showed that the relative abundances of OTUs could be markedly changed depending on the developmental stage, the location within the gut, and the honey bee species. The possibility of using the gut bacterial community as an indicator of honey bee health was discussed.     

Molecular characterization and tissue localization of sensory neuron membrane protein from Chinese honey bee, Apis cerana cerana (Hymenoptera: Apidae)

Sensory neuron membrane protein (SNMP) is an olfactory receptor with photoaffinity analogs, capable of binding the pheromone membrane protein receptor deduced from receptor membrane protein with the pheromone–pheromone binding protein complex. However, this hypothesis has not yet been experimentally verified. In this experiment, the cDNA sequence encoding an open reading frame (ORF) of the SNMP gene AccSNMP1 (GenBank, KC012595) was cloned from Chinese honey bee, Apis cerana cerana Fabricius. Results from sequence analysis showed that this gene is 1,563 bp long, and that the ORF encodes 520 amino acids with a predicted molecular weight of 58.02 kDa, and has a theoretical isoelectric point of 5.83. Furthermore, there are two putative transmembrane domains. Multiple sequence alignment indicated that the AccSNMP1 gene from A. cerana cerana had different degrees of identity with the corresponding genes in nineteen other insects at the amino acid level. Phylogenetic analysis of the aligned sequences showed that A. cerana cerana is closely related to Apis mellifera Linnaeus and Bombus impatiens Cresson. Its distribution in tissues, as quantified using real-time RT-PCR, indicated that AccSNMP1 is highly expressed in the antennae and legs of A. cerana cerana, and there was a significant difference (p < 0.05) in gene expression between those tissues and tissues in the thorax, abdomen, snout, and head (not including antennae). Western blotting also confirmed the existence in the antennae of AccSNMP1 with an M _W of 58.0 kDa, which is the same as the expected value of 58.02 kDa. An immunohistochemistry study showed that AccSNMP1 is expressed in the trichoid sensilla of A. cerana cerana antenna. Therefore, the results of this study provide the basis for further studies of the function of SNMP from A. cerana cerana.     

A SNP Based High-Density Linkage Map of Apis cerana Reveals a High Recombination Rate Similar to Apis mellifera

Background

The Eastern honey bee, Apis cerana Fabricius, is distributed in southern and eastern Asia, from India and China to Korea and Japan and southeast to the Moluccas. This species is also widely kept for honey production besides Apis mellifera. Apis cerana is also a model organism for studying social behavior, caste determination, mating biology, sexual selection, and host-parasite interactions. Few resources are available for molecular research in this species, and a linkage map was never constructed. A linkage map is a prerequisite for quantitative trait loci mapping and for analyzing genome structure. We used the Chinese honey bee, Apis cerana cerana to construct the first linkage map in the Eastern honey bee.

Results

F2 workers (N = 103) were genotyped for 126,990 single nucleotide polymorphisms (SNPs). After filtering low quality and those not passing the Mendel test, we obtained 3,000 SNPs, 1,535 of these were informative and used to construct a linkage map. The preliminary map contains 19 linkage groups, we then mapped the 19 linkage groups to 16 chromosomes by comparing the markers to the genome of A. mellfiera. The final map contains 16 linkage groups with a total of 1,535 markers. The total genetic distance is 3,942.7 centimorgans (cM) with the largest linkage group (180 loci) measuring 574.5 cM. Average marker interval for all markers across the 16 linkage groups is 2.6 cM.

Conclusion

We constructed a high density linkage map for A. c. cerana with 1,535 markers. Because the map is based on SNP markers, it will enable easier and faster genotyping assays than randomly amplified polymorphic DNA or microsatellite based maps used in A. mellifera.     

Genome-wide characterization of long intergenic non-coding RNAs (lincRNAs) provides new insight into viral diseases in honey bees Apis cerana and Apis mellifera

Background

Long non-coding RNAs (lncRNAs) are a class of RNAs that do not encode proteins. Recently, lncRNAs have gained special attention for their roles in various biological process and diseases.

Results

In an attempt to identify long intergenic non-coding RNAs (lincRNAs) and their possible involvement in honey bee development and diseases, we analyzed RNA-seq datasets generated from Asian honey bee (Apis cerana) and western honey bee (Apis mellifera). We identified 2470 lincRNAs with an average length of 1011 bp from A. cerana and 1514 lincRNAs with an average length of 790 bp in A. mellifera. Comparative analysis revealed that 5 % of the total lincRNAs derived from both species are unique in each species. Our comparative digital gene expression analysis revealed a high degree of tissue-specific expression among the seven major tissues of honey bee, different from mRNA expression patterns. A total of 863 (57 %) and 464 (18 %) lincRNAs showed tissue-dependent expression in A. mellifera and A. cerana, respectively, most preferentially in ovary and fat body tissues. Importantly, we identified 11 lincRNAs that are specifically regulated upon viral infection in honey bees, and 10 of them appear to play roles during infection with various viruses.

Conclusions

This study provides the first comprehensive set of lincRNAs for honey bees and opens the door to discover lincRNAs associated with biological and hormone signaling pathways as well as various diseases of honey bee.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1868-7) contains supplementary material, which is available to authorized users.     

Differential hygienic behavior of Apis cerana F. and Apis mellifera L. to Sacbrood virus infection

Beekeeping with Apis cerana of Korean apiculture is facing with serious colony collapse caused by invasive Sacbrood virus (SBV) disease. This fatal brood disease was the main reason of more than 90% colony lost in Korea leading almost the extinct crisis. Sacbrood virus can infect either larvae or adult honeybees, with a higher sensibility of larvae to the infection. Since SBV has spread to all over the country, efforts have been made to treat and prevent this devastating disease although no effective results have so far been obtained. Several studies have demonstrated that Apis mellifera bee colonies that express an efficient hygienic behavior exhibit a higher resistance to the brood disease. In this study we demonstrated that the differences of hygienic behavior between A. cerana and A. mellifera. A. cerana more efficiently removed the pin-killed brood than A. mellifera. On the other hand, A. mellifera more efficiently removed SBV-infected larvae and SBV-dead brood than A. cerana. However, it remains unclear whether the advantage of hygienic bee could have efficacy against Sacbrood disease on A. cerana colonies.     

Proboscis behavioral response of four honey bee Apis species towards different concentrations of sucrose,glucose, and fructose

Honey bees forage for pollen and nectar. Sugar is an important stimulus for foraging and a major source of energy for honey bees. Any differential response of bees to different concentrations of sugary nectar can affect their foraging. The sugar responsiveness of Apis species (Apis dorsata, Apis florea, and Apis cerana) was determined in comparison to that of Apis mellifera by evaluating the proboscis extension response (PER) with eight serial concentrations (0.00001, 0.0001, 0.001, 0.01, 0.1, 0.5, 1.0, and 1.5 M) of sucrose, glucose and fructose. Nectar foragers of bee species (A. dorsata, A. florea, A. cerana, and A. mellifera) exhibited an equal response for sucrose, glucose, and fructose, with no significant differences in their PER at all tested concentrations of these sugars within the same species. The inter-species comparison between Apis species revealed the differential responsiveness to the different concentrations of sugars, and the lowest concentration at which a response occurs was considered as the response threshold of these bee species for sugar solutions. A. mellifera presented significantly higher responsiveness than A. dorsata to low concentrations (0.00001, 0.0001, 0.001, 0.01, and 0.1 M) of sucrose, glucose and fructose. A. mellifera displayed a significantly higher response to water than A. dorsata. A. florea and A. mellifera presented no significant difference in their responsiveness to sucrose, glucose, and fructose at all tested concentrations, and their water responsiveness was also significantly at par but relatively higher in A. mellifera than in A. florea. Likewise, the responsiveness of A. cerana and A. mellifera to different concentrations of sucrose, glucose and fructose was significantly at par with no difference in their water responsiveness. This study represents preliminary research comparing the response of different honey bee species to three sugar types at different concentrations. The results imply that the native species are all better adapted than A. mellifera under local climate conditions.     

Hemolymph proteome changes during worker brood development match the biological divergences between western honey bees (Apis mellifera) and eastern honey bees (Apis cerana)

Background

Hemolymph plays key roles in honey bee molecule transport, immune defense, and in monitoring the physiological condition. There is a lack of knowledge regarding how the proteome achieves these biological missions for both the western and eastern honey bees (Apis mellifera and Apis cerana). A time-resolved proteome was compared using two-dimensional electrophoresis-based proteomics to reveal the mechanistic differences by analysis of hemolymph proteome changes between the worker bees of two bee species during the larval to pupal stages.

Results

The brood body weight of Apis mellifera was significantly heavier than that of Apis cerana at each developmental stage. Significantly, different protein expression patterns and metabolic pathways were observed in 74 proteins (166 spots) that were differentially abundant between the two bee species. The function of hemolymph in energy storage, odor communication, and antioxidation is of equal importance for the western and eastern bees, indicated by the enhanced expression of different protein species. However, stronger expression of protein folding, cytoskeletal and developmental proteins, and more highly activated energy producing pathways in western bees suggests that the different bee species have developed unique strategies to match their specific physiology using hemolymph to deliver nutrients and in immune defense.

Conclusions

Our disparate findings constitute a proof-of-concept of molecular details that the ecologically shaped different physiological conditions of different bee species match with the hemolymph proteome during the brood stage. This also provides a starting point for future research on the specific hemolymph proteins or pathways related to the differential phenotypes or physiology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-563) contains supplementary material, which is available to authorized users.     

Comparative susceptibility and immune responses of Asian and European honey bees to the American foulbrood pathogen,Paenibacillus larvae

American foulbrood (AFB) disease is caused by Paenibacillus larvae. Currently, this pathogen is widespread in the European honey bee— Apis mellifera. However, little is known about infectivity and pathogenicity of P. lan'ae in the Asiatic cavity-nesting honey bees, Apis cerana. Moreover, comparative knowledge of P. larvae infectivity and pathogenicity between both honey bee species is scarce. In this study, we examined susceptibility, larval mortality, survival rate and expression of genes encoding antimicrobial peptides (AMPs) including defensin, apidaecin, abaecin, and hymenoptaecin in A. mellifera and A. cerana when infected with P. larvae. Our results showed similar effects of P. larvae on the survival rate and patterns of AMP gene expression in both honey bee species when bee larvae are infected with spores at the median lethal concentration (LC5 0 ) for A. mellifera. All AMPs of infected bee larvae showed significant upregulation compared with noninfected bee larvae in both honey bee species. However, larvae of A. cerana were more susceptible than A. mellifera when the same larval ages and spore concentration of P. larvae were used. It also appears that A. cerana showed higher levels of AMP expression than A. mellifera. This research provides the first evidence of survival rate, LC50 and immune response profiles of Asian honey bees, A. cerana, when infected by P. larvae in comparison with the European honey bee, A. mellifera.     

csd alleles in the red dwarf honey bee (Apis florea,Hymenoptera: Apidae) show exceptionally high nucleotide diversity

Abstract The single locus complementary sex determination (sl‐csd) gene is the primary gene determining the gender of honey bees (Apis spp.). While the csd gene has been well studied in the Western honey bee (Apis mellifera), and comparable data exist in both the Eastern honey bee (Apis cerana) and the giant honey bee (Apis dorsata), no studies have been conducted in the red dwarf honey bee, Apis florea. In this study we cloned the genomic region 3 of the A. florea csd gene from 60 workers, and identified 12 csd alleles. Analysis showed that similar to A. mellifera, region 3 of the csd gene contains a RS domain at the N terminal, a proline‐rich domain at the C terminal, and a hypervariable region in the middle. However, the A. florea csd gene possessed a much higher level of nucleotide diversity, compared to A. mellifera, A. cerana and Apis dorsata. We also show that similar to the other three Apis species, in A. florea, nonsynonymous mutations in the csd gene are selectively favored in young alleles.     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

Transcriptome differences in the hypopharyngeal gland between Western Honeybees (Apis mellifera) and Eastern Honeybees (Apis cerana)

Background

Apis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager).

Results

Twelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs.

Conclusions

In this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-744) contains supplementary material, which is available to authorized users.     

Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera)

Nosema ceranae, a microsporidian parasite originally described in the Asian honey bee Apis cerana, has recently been found to be cross-infective and to also parasitize the European honey bee Apis mellifera. Since this discovery, many studies have attempted to characterize the impact of this parasite in A. mellifera honey bees. Nosema species can infect all colony members, workers, drones and queens, but the pathological effects of this microsporidium has been mainly investigated in workers, despite the prime importance of the queen, who monopolizes the reproduction and regulates the cohesion of the society via pheromones. We therefore analyzed the impact of N. ceranae on queen physiology. We found that infection by N. ceranae did not affect the fat body content (an indicator of energy stores) but did alter the vitellogenin titer (an indicator of fertility and longevity), the total antioxidant capacity and the queen mandibular pheromones, which surprisingly were all significantly increased in Nosema-infected queens. Thus, such physiological changes may impact queen health, leading to changes in pheromone production, that could explain Nosema-induced supersedure (queen replacement).     

Imidacloprid Alters Foraging and Decreases Bee Avoidance of Predators

Concern is growing over the effects of neonicotinoid pesticides, which can impair honey bee cognition. We provide the first demonstration that sublethal concentrations of imidacloprid can harm honey bee decision-making about danger by significantly increasing the probability of a bee visiting a dangerous food source. Apis cerana is a native bee that is an important pollinator of agricultural crops and native plants in Asia. When foraging on nectar containing 40 µg/L (34 ppb) imidacloprid, honey bees (Apis cerana) showed no aversion to a feeder with a hornet predator, and 1.8 fold more bees chose the dangerous feeder as compared to control bees. Control bees exhibited significant predator avoidance. We also give the first evidence that foraging by A. cerana workers can be inhibited by sublethal concentrations of the pesticide, imidacloprid, which is widely used in Asia. Compared to bees collecting uncontaminated nectar, 23% fewer foragers returned to collect the nectar with 40 µg/L imidacloprid. Bees that did return respectively collected 46% and 63% less nectar containing 20 µg/L and 40 µg/L imidacloprid. These results suggest that the effects of neonicotinoids on honey bee decision-making and other advanced cognitive functions should be explored. Moreover, research should extend beyond the classic model, the European honey bee (A. mellifera), to other important bee species.     

Nosema ceranae in European honey bees (Apis mellifera)

Nosema ceranae is a microsporidian parasite described from the Asian honey bee, Apis cerana. The parasite is cross-infective with the European honey bee, Apis mellifera. It is not known when or where N. ceranae first infected European bees, but N. ceranae has probably been infecting European bees for at least two decades. N. ceranae appears to be replacing Nosema apis, at least in some populations of European honey bees. This replacement is an enigma because the spores of the new parasite are less durable than those of N. apis. Virulence data at both the individual bee and at the colony level are conflicting possibly because the impact of this parasite differs in different environments. The recent advancements in N. ceranae genetics, with a draft assembly of the N. ceranae genome available, are discussed and the need for increased research on the impacts of this parasite on European honey bees is emphasized.     

The pheromones of laying workers in two honeybee sister species: <Emphasis Type="Italic">Apis cerana</Emphasis> and <Emphasis Type="Italic">Apis mellifera</Emphasis>

When a honeybee colony loses its queen, workers activate their ovaries and begin to lay eggs. This is accompanied by a shift in their pheromonal bouquet, which becomes more queen like. Workers of the Asian hive bee Apis cerana show unusually high levels of ovary activation and this can be interpreted as evidence for a recent evolutionary arms race between queens and workers over worker reproduction in this species. To further explore this, we compared the rate of pheromonal bouquet change between two honeybee sister species of Apis cerana and Apis mellifera under queenright and queenless conditions. We show that in both species, the pheromonal components HOB, 9-ODA, HVA, 9-HDA, 10-HDAA and 10-HDA have significantly higher amounts in laying workers than in non-laying workers. In the queenright colonies of A. mellifera and A. cerana, the ratios (9-ODA)/(9-ODA + 9-HDA + 10-HDAA + 10-HDA) are not significantly different between the two species, but in queenless A. cerana colonies the ratio is significant higher than in A. mellifera, suggesting that in A. cerana, the workers’ pheromonal bouquet is dominated by the queen compound, 9-ODA. The amount of 9-ODA in laying A. cerana workers increased by over 585% compared with the non-laying workers, that is 6.75 times higher than in A. mellifera where laying workers only had 86% more 9-ODA compared with non-laying workers.